A number of peptides and proteins have been developed as useful drugs largely by genetic engineering technique. However, their therapeutic potential is often severely limited due to their intrinsic properties as peptides or proteins. These molecules are readily metabolized by proteases and rapidly excreted by renal filtration. Furthermore, they may induce immunological reactions even if their sequences are similar to human proteins. It is therefore desirable to develop biologically more stable and long acting peptide- or protein-based drugs by chemical modification.
Polyethylene glycol (PEG) is a hydrophilic, biocompatible and non-toxic water-soluble polymer of general formula H—(OCH2CH2)n—OH, wherein n>4. Its molecular weight varies from 300 to 40,000 Daltons.
It has been demonstrated that the conjugation of PEG to proteins and/or peptides significantly increases their duration of biological activity. PEG provides a more stable conformation and increases the size of the molecule, thus reducing its metabolic degradation and renal clearance. Furthermore, PEGylation may reduce the immunogenecity and improve the solubility of the peptide or the protein.
Various PEG-protein conjugates were found to be protected from proteolysis and/or to have a reduced immunogenicity [Monfardini, et al., Biocon. Chem., 6, 62–69 (1995); and Yamsuki et al., Agric. Biol. Chem., 52, 2185–2196 (1988)]
PEG conjugation is an already established methodology for peptide and protein modification pioneered by the work of Davis and Abuchowski [Abuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol. Chem., 252, 3582 (1977)]. PEG conjugation to peptides or proteins generally involved the activation of PEG and coupling of the activated PEG-intermediates directly to target proteins/peptides or to a linker, which is subsequently activated and coupled to target proteins/peptides. One of the key issues with the conjugation is the chemistry used to activate PEG and PEG-linker, which in turn will determine the coupling efficiency and specificity of the activated PEG or PEG-linker to its targets.
For example, the trichlorotriazine—activated PEG, which was found to be toxic and non-specific, was later on replaced by various activated PEG that could react specifically to amino group of the target peptides or proteins [Bencham C. O. et al., Anal. Biochem., 131, 25 (1983); Veronese, F. M. et al., Appl. Biochem., 11, 141 (1985).; Zalipsky, S. et al., Polymeric Drugs and Drug Delivery Systems, adrs 9–110 ACS Symposium Series 469 (1999); Zalipsky, S. et al., Europ. Polym. J., 19, 1177–1183 (1983); Delgado, C. et al., Biotechnology and Applied Biochemistry, 12, 119–128 (1990)], to sulfhydryl group [Sartore, L., et al., Appl. Biochem. Biotechnol., 27, 45 (1991); and Morpurgo et al., Biocon. Chem., 7, 363–368 (1996)] or to guanidine residues [Pande, C. S. et al., Proc.Nat.Acad.Sci., 77, 895–899 (1980)]. Another technical difficulty in protein PEGylation arises from the fact that a protein or a peptide often contains not only different functional groups such as amino, guanidine, hydroxyl or sulfhydryl, but it also has more than one of them. As a result, the product contains a mixture of conjugates with different PEG-protein stochiometries. The conjugation of PEG to growth hormone (GH) represents a typical example of such problem [Clark, R. et al., J. Biol. Chem., 36, 21969–21977 (1996)]. It was demonstrated that Lys residues of GH were PEGylated at random positions. Site-specific PEGylation of a protein remains a chemical challenge.
To increase the coupling specificity of the PEG to a protein or peptide, without significantly reducing its coupling efficiency, the carbonate esters of PEG have been frequently used to form the PEG-peptide or PEG-protein conjugates. N,N′-disuccinimidylcarbonate (DSC) has been used in the reaction with PEG to form active mixed PEG-succinimidyl carbonate that was subsequently reacted with a nucleophilic group of a linker or an amino group of a target protein/peptide (U.S. Pat. Nos. 5,281,698 and 5,932,462). In a similar type of reaction, 1,1′-(dibenzotriazolyl)carbonate and di-(2-pyridyl)carbonate have been reacted with PEG to form PEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No. 5,382,657), respectively.
However, the carbonate esters are not easily prepared. In general, to achieve good yield, phosgene is often used in their preparation. This reagent can be difficult to handle and dangerous in the large-scale synthesis.. Thus, the use of other but similar active esters, such as N,N′-disuccinimidyl oxalate and 1,1′-bis[6-(trifluoromethy)benzo-triazolyl] oxalate, which are prepared from oxalyl chloride, may be superior and safer in the preparation of PEG mixed carbonate active esters. Furthermore, the oxalate active esters can be more economically prepared than the carbonate active ester.